(416f) Investigating Cholera Toxin Binding Mechanism with Gangliosides Via Kinetic Modeling and Experimental Measurements

Cholera toxin (CTx), which is secreted by Vibrio cholera, is a toxin protein that is the main culprit of cholera. CTX is an AB₅ toxin, containing an enzymatic A-subunit and five identical cholera toxin B-subunits (CTB) [1]. CTB binds the gangliosides such as GM1 on host cell membranes leading to the endocytosis of CTx. In cytoplasm, the toxic A-subunit adversely increases the intracellular cyclic AMP level in the host cell [2]. The elevated cyclic AMP concentration results in severe fluid loss, which can be life-threatening if persists. In order to better cope with CTx invasion, the detailed mechanism of CTB-GM1 interactions, which is the prerequisite for CTx cell entry, needs to be elucidated. It is worth noting that a pentavalent CTB can simultaneously binds to multiple copies of GM1 in host cell membranes. The positive cooperativity between each binding subunit can further enhance the overall binding avidity [1, 3]. The conventional ligand-receptor analysis (monovalent binding model) is not sufficient to describe the complex multivalent CTx binding mechanisms. In this study, both in silico and in vitro approaches are employed to investigate complex interactions between CTB and GM1 in a cell-membrane-mimicking environment.

Lauer et al. developed a five-step kinetic model for CTB binding to GM1 on lipid bilayer [3]. However, the model did not account for the aforementioned positive cooperativity among CTB subunits. Here, we integrated the five-step model with other binding mechanism proposed by Turnbull et al. [1]. The new kinetic model incorporated association/dissociation constants with cooperativity factors. Based on the number of bound GM1 per CTB and the local configuration, seven CTB-GM1 complexes were considered in our simulation. The accuracy of kinetic model were verified by a novel protein binding assay. We used a quantitative nanocube-based biosensor to collect CTB-GM1 binding kinetics in a cell membrane mimicking environment [5]. The high-throughput and easy-to-use features of nanocube sensor allow collect massive data at various experimental conditions. In addition, we applied the same methodology to examine other host cell receptors such as GM2 and fucosyl-GM1, which exhibit distinct CTB binding kinetics. In summary, we integrated theoretical modeling and experimental measurements to explore cholera pathogenesis. Our work offers a systematic tool for the biomedical community to reveal the pathogenesis of other diseases.

Reference

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